The present invention relates generally to methods and devices for the quantitative determination of the presence of an enzyme in a sample by means of an analyte catalyzed reaction to form a reaction product. More specifically, the invention relates to methods wherein the analyte enzyme is immobilized at a reaction site on a chromatographic medium and substrates and cofactors for the analyte catalyzed reaction and products of that reaction are transported to and from the site by means of chromatographic solvent transport.
Methods known in the art for the detection of enzyme analytes in a sample generally involve contacting the sample to be analyzed with a mixture of substrate and cofactor materials the reaction of which is catalyzed by the analyte enzyme. The presence of the analyte in the sample may be determined by observation of the rate of production of a reaction product or consumption of a reactant (substrate or cofactor) as a consequence of the analyte-catalyzed reaction. Where the rate of production of a product of the reaction is used to indicate the presence of an enzyme, the product may be detected visually or spectrophotometrically. Alternatively, where a reaction product is not readily detectable by visual or spectrophotometric means, it may be detected by being subjected to one or more subsequent reactions which yield a readily detectable reaction product. Such reactions frequently involve activation of a dye precursor material. Where the rate of consumption of a reactant is used to indicate the presence of an enzyme, that reactant should be detectable visually or spectrophotometrically. A commonly used reactant is the cofactor nicotine adenine dinucleotide (NADH) which is detectable spectrophotometrically (at 340 nm) or fluorometrically (at 410 nm). The cofactor is oxidized in many enzyme catalyzed reactions to NAD.sup.+ which does not emit the characteristic spectrophotometric or fluorometric signal. Many analyte catalyzed reactions are therefore followed by tracking the disappearance of NADH.
For example, methods are known for the detection of the enzyme alanine aminotransferase (ALT) increased blood levels of which are associated with hepatitis. Of interest to the present invention is the disclosure of Murray, Methods in Clinical Chemistry, pp. 1062-1065, Pesce & Waplan, eds., Mosby Publishing Co., St. Louis, Mo. (1987). ALT catalyzes the transamination reaction of L-alanine with alpha-ketoglutarate to produce pyruvate and L-glutamate. According to one widely used procedure for the detection of ALT, serum is incubated with L-alanine and alpha-ketoglutarate and after a measured length of time the reaction is stopped and the newly formed pyruvate is reacted with dinitrophenylhydrazine (DNPH) to form the corresponding hydrazone. The reaction mixture is then alkalinized to produce a blue color caused by the anion form of the hydrazone. The colorimetric procedure suffers from limited linearity as a consequence of feedback inhibition of the ALT by pyruvate. According to another procedure, NADH is incorporated in the reaction medium as is lactate dehydrogenase. The lactate dehydrogenase catalyzes the conversion of pyruvate to lactate with the simultaneous oxidation of reduced NADH to oxidized NAD.sup.+. The disappearance of NADH is followed spectrophotometrically or fluorometrically.
Similar methods are known for the detection of the enzyme aspartate aminotransferase (AST) increased blood serum levels of which are associated with acute myocardial infarction, acute pancreatitis, viral and toxic hepatitis and acute cirrhosis. AST catalyzes the transamination reaction of aspartate and alpha-ketoglutarate to oxaloacetate and glutamate. Methods for the detection of this enzyme involve incubation of a sample to be tested with a solution containing aspartate, alpha-ketoglutarate and 2,4-dinitrophenylhydrazine such that the AST catalyzed production of oxaloacetate is coupled with the formation of a 2,4-dinitrophenyl-hydrazone-derivative which absorbs light at 520 nm. The presence of AST in the sample fluid is thus indicated by a color signal which can be measured spectrophotometrically or may be compared with a color chart to provide a semi-quantitative indication of the presence of the AST. Similar procedures are known where the 2,4-dinitrophenylhydrazone dye precursor is replaced by an azozene dye which is capable of reacting with oxaloacetic acid. Still other methods for AST detection have become known involving the conversion of oxaloacetate to malate in a reaction utilizing malate dehydrogenase with NADH and NAD.sup.+. Such analytical reactions may be carried out in containers such as test tubes and microtitre wells but may also be carried out on absorbent dip strips.
Of interest to the present invention is the disclosure of Forgione, U.S. Pat. No. 3,875,014 which discloses test indicators for the determination of AST concentrations in sera utilizing aspartic acid, alpha-ketoglutaric acid and a diazonium salt according to the reactions disclosed above. The test indicator comprises a pair of porous strips, adhered to each other with an adhesive which is selectively permeable to oxaloacetic acid, the first of which comprises the substrates L-aspartic acid and alpha-ketoglutaric acid. The second comprises a dried diazonium salt. The indicator is contacted with sera which, if it contains AST, catalyzes the reaction of the substrates to form oxaloacetic acid. Any oxaloacetic acid formed thereby then diffuses to the second strip and activates a color reaction with the diazonium salt.
The various assay methods for the quantitative detection of enzyme analytes tend to be limited in their accuracy by the nature of the kinetics of the enzyme catalyzed reaction. Such assays typically contact a sample containing an unknown amount of enzyme with substrates for that enzyme and determine the quantity of product produced by that reaction over a given period. The amount of product is indicative of the average rate of reaction which is itself related to the quantity of enzyme in the sample. The use of average rates of reaction to determine the quantity of enzyme present is limited by the fact that under typical assay conditions, such reactions do not generally have constant reaction rates. Enzyme catalyzed reactions carried out in a fixed volume of substrate/cofactor solution are affected by a number of startup and concentration effects which affect the rate of reaction. Typically, enzyme catalyzed reactions are characterized by a low start-up rate before reaching a "steady state." As the reaction proceeds and members of the enzyme substrate/cofactor group are consumed and their concentration diminishes, the reaction rate will slow. The rate of reaction will also be retarded as a consequence of feedback inhibition by accumulation of reaction products. Where the analyte catalyzed reaction is terminated by a change in reaction conditions or addition of an inhibitor, cessation of the reaction may not be entirely instantaneous thus adding additional uncertainty into the determination of average reaction kinetics. The true steady state reaction kinetics of the analyte catalyzed reaction may therefore vary significantly from the average reaction rate indicated by evaluation over a finite time period. Determinations of enzyme concentrations based on determinations of average reaction rates will thus be inaccurate to the degree that steady state reaction kinetics differ from average reaction rates. It is therefore desired to produce an assay method capable of evaluating the steady state reaction kinetics of a given reaction and preferably the instantaneous kinetics at any time.